The biosynthesis of many clinically used antibiotics and antitumor compounds occurs on large enzymes (100-1600 kDa) known to have complex arrays of covalent intermediates, ideal for study by mass spectrometry (MS). With 10-20 biosynthetic intermediates bound to these polyketide (PKS) and non-ribosomal peptide synthetases (NRPSs) via acid-stable thioester linkages, natural product assembly is modeled to occur in a "conveyor belt" style. Using state-of-the-art MS techniques and a custom Quadrupole-Fourier-Transform hybrid instrument, we will interrogate biosynthetic intermediates with unprecedented detail to dissect the kinetic timing, substrate specificities, and editing functions for natural and engineered NRPS and hybrid NRPS-PKS systems. For the first time, the percent occupancy of intermediates at many carrier sites will be correlated with one another to access the fundamental biochemistry of NRPS and PKSs and decipher between a "crawling" vs. a "fast translocation" model of how these enzymes function. NRPS and NRPS/PKS hybrid systems involved in production of yersiniabactin, epothilone, and gramicidin S will be studied in vitro, with fast purification from whole cells under acidic conditions also planned to monitor covalent states of specific carrier sites in vivo. Through determination of relative processing rates for mixtures of substrates in competition experiments, enzymes will be evaluated via efficient generation of structure-activity relationships to facilitate future engineering of these and other NRPS-PKS hybrid systems. To help overcome editing capabilities, such direct and efficient detection of enzyme-bound intermediates is most critical in the earliest stages of the NRPS/PKS engineering process, where substrate selectivity's are near maximal and single turnover can be the norm. Thus, the unique information produced from these studies will illuminate mechanistic details, prove the existence of putative intermediates, and facilitate the design of nonnatural enzymes for (combinatorial) generation of new bioactive compounds by NRPS and PKSs.